The increasing ease of cloning and synthesizing DNA sequences has greatly expanded opportunities for detecting particular nucleic acid sequences of interest. No longer must one rely on the use of immunocomplexes for the detection of pathogens, ligands, antigens, and the like. Rather than detecting particular determinant sites, one can detect DNA sequences or RNA sequences associated with a particular cell. In this manner, diseases can be diagnosed, phenotypes and genotypes can be analyzed, as can polymorphisms, relationships between cells, and the like.
Analyses of DNA sequences typically involve the binding of an analyte sequence to a solid support and hybridization of a complementary sequence to the bound sequence. The annealing and complexing steps usually involve an extended period of time and require careful washing to minimize non-specific background signals. Applicants' co-pending application Ser. No. 807,624, describes new techniques for analyzing nucleic acid sequences which are faster, minimize the number of manipulative steps, and provide for an increased signal to noise ratio. This application, a continuation-in-part of Ser. No. 807,624, the disclosure of which is incorporated by reference herein, is directed in particular to novel polynucleotide probes useful, inter alia, in the techniques described in applicants' co-pending parent application.
The majority of polynucleotide probes in current use are radioactively labeled, e.g. with isotopes of hydrogen (.sup.3 H), phosphorus (.sup.32 P), carbon (.sup.14 C) or iodine (.sup.125 I). These materials are relatively simple to synthesize by direct inclusion of the radioactive moieties, e.g. by kinasing with .sup.32 P-labeled ATP, equilibrating with tritiated water, or the like. As is well known, however, use of such radioactive labels has drawbacks, and other detectable species which are not radioactive are preferred.
In order to incorporate other, non-radioactive types of detectable species in a nucleotide, some sort of chemical modification of the nucleotide is required. It is widely recognized that nucleotide modification is a difficult and sensitive procedure, as any modification reaction has to be mild enough to leave the RNA or DNA molecules intact, while giving a modified nucleotide product which can participate in normal base pairing and stacking interactions. These considerations typically limit nucleotide substitution positions to the 5-position of a pyrimidine and the 8-position of a purine, as noted in the literature (see, e.g., European patent application Ser. No. 063879, cited supra).
Other considerations must also be taken into account. Base pairing may be hindered during hybridization if the detectable label is at one end of the nucleotide chain rather than present at some point within it. Further, it has proved difficult to provide even non-radioactively labeled probes which may be inexpensively synthesized in large quantity. Thus, many known probes are limited in their potential applications.